CN103189509A - Method for detecting nucleic acid, method for optically observing sample, and phosphor - Google Patents

Method for detecting nucleic acid, method for optically observing sample, and phosphor Download PDF

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Publication number
CN103189509A
CN103189509A CN2011800532046A CN201180053204A CN103189509A CN 103189509 A CN103189509 A CN 103189509A CN 2011800532046 A CN2011800532046 A CN 2011800532046A CN 201180053204 A CN201180053204 A CN 201180053204A CN 103189509 A CN103189509 A CN 103189509A
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fluorescence
nucleic acid
copper
sample
spectrum
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新田尚
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Sony Corp
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Sony Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/12—Circuits of general importance; Signal processing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Abstract

[Problem] To provide a method capable of detecting a nucleic acid with which a nucleic acid can be simply detected particularly without requiring a complicated operation such as mixing of a liquid or washing in a microscale flow path or the like. [Solution] Provided is a method for detecting a nucleic acid, comprising a step of bringing a sample containing a nucleic acid into contact with copper and a step of detecting fluorescence emitted from the sample. According to the method for detecting a nucleic acid, merely by bringing a sample containing a nucleic acid into contact with copper, fluorescence derived from a complex between the nucleic acid and copper can be simply detected.

Description

Optical observation method and the fluorescent substance of the detection method of nucleic acid, sample
Technical field
Present technique relates to the detection method of nucleic acid, optical observation method and the fluorescent substance of sample.More specifically, present technique relates to the detection method of nucleic acid and the optical observation method of sample, two kinds of methods all be based on from nucleic acid that copper contacts the fluorescence that sends, and the fluorescent substance that comprises copper and nucleic acid.
Background technology
In recent years, in a plurality of fields that comprise medical field, medicament research and development field, clinical examination field, field of food, agriculture field, engineering field, forensic science and criminal evaluation field, extensively carry out the technical study that utilizes nucleic acid.Recently, technological development chip lab (lab-on-chip), and be applied to be arranged on the dyeing of carrying out nucleic acid in the micro-scale stream in the microchip, detection, amplification etc.
As the basic fundamental that detects nucleic acid, a kind of method that adopts high-visibility pigment dyeing nucleic acid is arranged.Known many high-visibility pigments, for example hoechst33342, DAPI, ethidium bromide, SYBR are green etc.For instance, hoechst33342 and DAPI are used for the nucleic acid staining of cell or tissue in flow cytometer or microscope.Ethidium bromide usually is used for the nucleic acid molecule dyeing to electrophoresis.The green grade of SYBR also is used in the nucleic acid amplification technologies of for example polymerase chain reaction the amplification procedure of real time nucleic acid detection.
Relevant with present technique, the known usually autofluorescence that is shown by cell when Fluirescence observation will be described.One of fluorescence is under the condition that copper exists, by the shown orange autofluorescence of the cell of UV-irradiation.For example, it is reported when adding copper the cell of the privileged site in drosophila larvae midgut emission fluorescent orange (with reference to non-patent document 1 to 8).In the drosophila larvae midgut, the cell of observing especially strong fluorescent orange is referred to as " copper cell " etc.It is reported, when the concentration when increasing of copper adding, observe fluorescence at the whole body wall (with reference to non-patent document 2) of the pericellular cell of copper (with reference to non-patent document 4) and larva.
Tenuigenin and the nucleus described at cell all can be observed above-mentioned fluorescent orange, and, especially in cytoplasmic particle, significantly detected (with reference to non-patent document 2～4,7).The wavelength region of having described fluorescence is between 590nm～630nm, and peak wavelength is 610nm, and the maximum excitation wavelength is that 340nm(is with reference to non-patent document 3).
For the organism except fruit bat, the autofluorescence with similar quality also is observed.For example, be reported in the rat experiment, by ultraviolet excitation (excitation wavelength is 310nm), in giving the individual liver of copper, observe fluorescent orange (peak wavelength with 605nm) (with reference to non-patent document 9).In addition, have and observe similar fluorescence (with reference to non-patent document 10) in the kidney that is reported in the rat model that in kidney and liver, has accumulated copper with age.Equally, have in the Liver Tissue (the non-patent document 12 of reference) that is reported in yeast (with reference to non-patent document 11) and Wilson patient and observe the autofluorescence with similar features.The Wilson disease be a kind of copper bar let out deficiency and in liver cell the accumulation copper genetic diseases.
Infer that the mixture (being abbreviated as " Cu-MT " hereinafter) of copper and metallothioneins (MT) is the fluorescent substance (with reference to non-patent document 14～23) of above-mentioned emission fluorescent orange.Cu-MT has such as the excitation wavelength of the 305nm in the non-patent document 13 and the wavelength of fluorescence of 565nm, and the wavelength characteristic of the wavelength of fluorescence of the excitation wavelength of the 310nm in the non-patent document 17 and 570nm.What can expect is that Cu-MT comprises univalent copper ion (Cu(I)) (with reference to non-patent document 13,15,17,19 and 23).
Interact and the mixture that contains pyrimidine or thiolate of emitting fluorescence is widely known by the people (with reference to non-patent document 24～29) as the fluorescent substance of cupric by pyrimidine or thiolate and copper.
On the other hand, the past has been studied the interaction of each metal ion species and nucleic acid.For example, known when monovalent copper ion and nucleic acid interaction, a little copper that comprises in the nucleus makes nucleic acid construct stable, but under the hydrogen peroxide coexistence, a little copper that comprises in the nucleus makes DNA impaired (with reference to non-patent document 30).Also have report, changed the absorption spectrum (with reference to non-patent document 30 and 31) of DNA with the interaction of copper.In addition, it is reported that the change of absorption spectrum depends on the base sequence of DNA (particularly, have the right polymer of G-C and contain the right polymer of A-T) (with reference to non-patent document 30).
The quoted passage tabulation
Non-patent document
Non-patent document 1:Physiological genetic studies on copper metabolism in the genus Drosophila. (1950) Genetics35,684-685
Non-patent document 2:Organization and function of the inorganic constituents of nuclei. (1952) Exp.Cell Res., Suppl.2:161-179
Non-patent document 3:Ultrastructure of the copper-accumulating region of the Drosophila larval midgut. (1971) Tissue Cell.3,77-102
Non-patent document 4:Specification of a single cell type by a Drosophila homeotic gene. (1994) Cell.76,689-702
Non-patent document 5:Two different thresholds of wingless signalling with distinct developmental consequences in the Drosophila midgut. (1995) EMBO J.14,5016-5026
Non-patent document 6:Calcium-activated potassium channel gene expression in the midgut of Drosophila. (1997) Comp.Biochem.Physiol.B Biochem.Mol.Biol.118,411-420
Non-patent document 7:Evidence that a copper-metallothionein complex is responsible for fluorescence in acid-secreting cells of the Drosophila stomach. (2001) Cell Tissue Res.304,383-389
Non-patent document 8:Peptidergic paracrine and endocrine cells in the midgut of the fruit fly maggot. (2009) Cell Tissue Res.336,309-323
Non-patent document 9:A luminescence probe for metallothionein in liver tissue:emission intensity measured directly from copper metallothionein induced in rat liver. (1989) FEBS Lett.257,283-286
Non-patent document 10:Direct visualization of copper-metallothionein in LEC rat kidneys:application of autofluorescence signal of copper-thiolate cluster. (1996) J.Histochem.Cytochem.44,865-873
Non-patent document 11:Incorporation of copper into the yeast Saccharomyces cerevisiae.Identification of Cu (I)--metallothionein in intact yeast cells. (1997) J.Inorg.Biochem.66,231-240
Non-patent document 12:Portmann B.Image of the month.Copper-metallothionein autofluorescence. (2009) Hepatology.50,1312-1313
Non-patent document 13:Luminescence properties of Neurospora copper metallothionein. (1981) FEBS Lett.127,201-203
Non-patent document 14:Copper transfer between Neurospora copper metallothionein and type3copper apoproteins. (1982) FEBS Lett.142,219-222
Non-patent document 15:Spectroscopic studies on Neurospora copper metallothionein. (1983) Biochemistry.22,2043-2048
Non-patent document 16:Metal substitution of Neurospora copper metallothionein. (1984) Biochemistry.23,3422-3427
Non-patent document 17:(Cu, Zn)-metallothioneins from fetal bovine liver.Chemical and spectroscopic properties. (1985) J.Biol.Chem.260,10032-10038
Non-patent document 18:Primary structure and spectroscopic studies of Neurospora copper metallothionein. (1986) Environ.Health Perspect.65,21-27
Non-patent document 19:Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants. (1988) J.Biol.Chem.263,6688-6694
Non-patent document 20:Luminescence emission from Neurospora copper metallothionein.Time-resolved studies. (1989) Biochem J.260,189-193
Non-patent document 21:Establishment of the metal-to-cysteine connectivities in silver-substituted yeast metallothionein (1991) J.Am.Chem.Soc.113,9354-9358
Non-patent document 22:Copper-and silver-substituted yeast metallothioneins:Sequential proton NMR assignments reflecting conformational heterogeneity at the C terminus. (1993) Biochemistry.32,6773-6787
Non-patent document 23:Luminescence decay from copper (I) complexes of metallothionein. (1998) Inorg.Chim.Acta.153,115-118
Non-patent document 24:Solution Luminescence of Metal Complexes. (1970) Appl.Spectrosc.24,319-326
Non-patent document 25:Fluorescence of Cu, Au and Ag mercaptides. (1971) Photochem.Photobiol.13,279-281
Non-patent document 26:Luminescence of the copper--carbon monoxide complex of Neurospora tyrosinase. (1980) FEBS Lett.111,232-234
Non-patent document 27:Luminescence of carbon monoxide hemocyanins. (1980) Proc.Natl.Acad.Sci.U.S.A.77,2387-2389
Non-patent document 28:Photophysical properties of hexanuclear copper (I) and silver (I) clusters. (1992) Inorg.Chem., 31,1941-1945
Non-patent document 29:Photochemical and photophysical properties of tetranuclear and hexanuclear clusters of metals with d10and s2electronic configurations. (1993) Acc.Chem.Res.26,220-226
Non-patent document 30:Interaction of copper (I) with nucleic acids. (1990) Int.J.Radiat.Biol.58,215-234
Non-patent document 31:Copper (I)-Catalyzed Regioselective " Ligation " of Azides and Terminal Alkynes. (2002) Ang.Chem.Int.Ed.41,2596-2599
Summary of the invention
The problem to be solved in the present invention
When adopting aforementioned existing fluorescent reagent to nucleic acid staining, require liquid reagent and sample mix, complicated thereby operation becomes.Especially, for the chip lab that carries out nucleic acid staining and detection in the micro-scale stream, it is very complicated that the production of chip, storage and use become.
A main purpose of present technique provides a kind of method that detects nucleic acid easily, need not complicated operations, for example mixing liquid and cleaning in the micro-scale stream.
The means of dealing with problems
In order to address the above problem, present technique provides a kind of method that detects nucleic acid, comprises step: the sample that contains nucleic acid is contacted with copper, and detect from the fluorescence of sample emission.According to the method for this detection nucleic acid, only by the sample that contains nucleic acid is contacted with copper, just can the easy fluorescence that detects the mixture that comes from nucleic acid and copper.In addition, based on detected fluorescence intensity and/or fluorescence spectrum, can provide the information about concentration, distribution or the shape of contained nucleic acid in the sample.
Especially, come from the fluorescence intensity of mixture of nucleic acid and copper and/or base sequence and the length that fluorescence spectrum depends on nucleic acid, and whether mispairing exists and changes in double-strandednucleic acid.Therefore, the method for this detection nucleic acid can be come mispairing in the two strands of the base sequence of analysis of nucleic acids and nucleic acid based on detected fluorescence intensity and/or fluorescence spectrum in detecting step.
In addition, the fluorescence that comes from nucleic acid and the mixture of copper in the uridylic is than the height in the cytosine(Cyt).Therefore, by this nucleic acid detection method, handle by hydrosulfate, make the non-methylated cytosine(Cyt) that comprises in the sample optionally be converted into uridylic, and determine the variable quantity of in detecting step detected intensity of fluorescence and/or spectrum, analyze thus in nucleic acid cytosine(Cyt) methylate or whether demethylation exists and measures, the position of cytosine(Cyt) or demethylation cytosine(Cyt) etc. methylates in base sequence.
In the detection method of this nucleic acid, copper can be solid.
In changing the detection method of nucleic acid, contact procedure preferably under the coexistence of salt, is implemented by the sample that contains nucleic acid is contacted with copper.And contact procedure is preferably by being that the rayed sample of 300 μ m～420 μ m is implemented to detect the fluorescence launch from sample with wavelength.
The technology of the present invention provides a kind of optical observation method of sample, may further comprise the steps: the sample that contains nucleic acid is contacted with copper, and detect from the fluorescence of sample emission.
In the optical observation method, sample can be cell.In this case, can provide information such as nuclear distribution about cell, position, quantity, size, shape.
In addition, present technique provides the fluorescent substance of the mixture that comprises cupric and nucleic acid.By base sequence and the length of the nucleic acid in the appropriate change mixture, can provide various fluorescent substances with different spectrum or intensity.
According to present technique, " nucleic acid " comprises natural acid (DNA and RNA).The for a change chemical structure of the chemical structure of ribose or phosphodiester bond (phosphodiester linkage) and the nucleic acid of the synthetic that provides in the natural acid by the people extensively are provided " nucleic acid ".The non-limiting example of synthetic nucleic acid comprises peptide nucleic acid(PNA) (RNA), thiophosphatephosphorothioate type oligonucleotide (S-oligos), bridge joint nucleic acid (BNA), lock nucleic acid (LNA) etc.
The invention effect
Present technique provides a kind of convenient detection method of nucleic acid, need not the complex operations such as mixing liquid and cleaning in the micro-scale stream.
Description of drawings
[Fig. 1] each graphic representation replaces accompanying drawing and is illustrated in fluorescence spectrum and the RFU value by making ssDNA contact with the CuSO4 of different concns and obtain under the condition that S.A. concentration is 50mM.(A) show fluorescence spectrum, (B) show RFU peak value (embodiment 1).
[Fig. 2] each graphic representation replaces accompanying drawing and is illustrated in fluorescence spectrum and the RFU value by making ssDNA contact with the CuSO4 of different concns and obtain under the condition that S.A. concentration is 50mM.(A) show fluorescence spectrum, (B) show RFU peak value (embodiment 1).
[Fig. 3] each graphic representation replaces accompanying drawing and is illustrated under the condition that S.A. concentration is 4mM by making oligomer DNA and concentration is fluorescence spectrum and the RFU value (embodiment 1) that the CuSO4 of 0.4mM contacts and obtains.
[Fig. 4] each graphic representation replaces accompanying drawing and is illustrated under the condition that S.A. concentration is 4mM by making oligomer DNA and concentration is fluorescence spectrum and the RFU value (embodiment 1) that the CuSO4 of 0.4mM contacts and obtains.
[Fig. 5] each graphic representation replaces accompanying drawing and is illustrated in CuSO4 concentration is that 0.4mM and S.A. concentration are at oligomer DNA (oligo-DNA) T(20 under the condition of 4mM), T(6) and the fluorescence spectrum that T(3) obtains and absorption spectrum (embodiment 1) over time.Above each to illustrate with RFU value (absolute value) be the fluorescence spectrum of ordinate zou, it is the fluorescence spectrum of ordinate zou that each of centre illustrates with RFU value (relative value), and following each illustrates absorption spectrum.
[Fig. 6] each graphic representation replaces accompanying drawing and is illustrated in CuSO4 concentration 0.4mM and S.A. concentration is at oligomer DNA T(20 under the condition of 4mM), T(6) and the fluorescence spectrum that T(3) obtains and absorption spectrum (embodiment 1) over time.(A) show the RFU peak value over time, when (B) showing wavelength and being 346nm over time.
[Fig. 7] each graphic representation replaces accompanying drawing also to be illustrated in oligomer DNA T(20), T(6) and the two-dimensional fluorescence spectrum that T(3) obtains.
[Fig. 8] graphic representation replaces accompanying drawing also to be illustrated in oligomer DNA T(20), T(6) and the excitation spectrum (dotted line) and the fluorescence spectrum (solid line) (embodiment 1) that T(3) obtain.
[Fig. 9] each graphic representation replaces accompanying drawing and is illustrated in the fluorescence spectrum of the oligomer DNA acquisition of three base length sequences that comprise that adenine and thymine is formed.
[Figure 10] each graphic representation replaces accompanying drawing and is illustrated in the highest RFU value (A) and the maximum FRU wavelength (B) (embodiment 1) of the fluorescence spectrum that the oligomer DNA of three base length sequences that comprise that adenine and thymine is formed obtains.
[Figure 11] each graphic representation replaces accompanying drawing and illustrates from comprising that sequence number is the fluorescence spectrum (embodiment 1) that obtains the oligomer DNA of SEQ ID NO:19 and 20.
[Figure 12] graphic representation replaces accompanying drawing and the fluorescence spectrum (embodiment 2) that obtains by the sample that contains ssDNA is contacted with solid copper is shown.
[Figure 13] graphic representation replaces accompanying drawing and illustrates by making the sample that contains ssDNA contact the fluorescence spectrum (embodiment 2) that obtains with the solid-state copper with different concns.
[Figure 14] each graphic representation replaces accompanying drawing and illustrates by the reaction soln that makes the sample that contains ssDNA and the salt that contains dissimilar or concentration contacting the fluorescence spectrum (embodiment 2) that obtains.
[Figure 15] each graphic representation replaces accompanying drawing also the ssDNA(A that contains different concns by making to be shown) or sample RNA(B) contact the fluorescence spectrum (embodiment 2) that obtains with solid copper.
[Figure 16] each graphic representation replaces accompanying drawing and illustrates by making to contain to have not that the sample of homotactic oligomer DNA contacts the fluorescence spectrum (embodiment 2) that obtains with solid copper.
[Figure 17] each graphic representation replaces accompanying drawing and illustrates by making to contain to have not that the sample of homotactic oligomer DNA contacts the fluorescence spectrum (embodiment 2) that obtains with solid copper.
[Figure 18] each graphic representation replaces accompanying drawing and illustrates by making to contain to have not that the sample of homotactic oligomer DNA contacts exciting-fluorescence spectrum (embodiment 2) of obtaining with solid copper.
[Figure 19] graphic representation replaces accompanying drawing and is illustrated in the fluorescence spectrum (embodiment 2) of the oligomer DNA acquisition of the composite sequence with 8-base cytosine(Cyt) and 12-base thymus pyrimidine.
[Figure 20] each graphic representation replaces accompanying drawing and is illustrated in the fluorescence spectrum (embodiment 2) of the double-stranded DNA acquisition that contains mispairing.
[Figure 21] each graphic representation replaces accompanying drawing and illustrates by the buffer type that changes reaction soln and the RFU value (embodiment 2) that the pH value obtains.
[Figure 22] each photo represents accompanying drawing and the fluoroscopic image (embodiment 3) that the copper that sputters on the glass surface is contacted with ssDNA and obtain is shown.
[Figure 23] each photo represents accompanying drawing and the fluoroscopic image (embodiment 3) that the copper that sputters on the glass surface is contacted with RNA and obtain is shown.
[Figure 24] graphic representation replaces accompanying drawing and the fluorescence intensity (embodiment 3) that the copper that sputters on the glass surface or silver is contacted with the sample that comprises DNA or RNA and obtain is shown.
[Figure 25] graphic representation replaces accompanying drawing and the fluorescence intensity that the copper that sputters on the glass surface contacted with ssDNA and obtain (embodiment 3) over time is shown.
[Figure 26] graphic representation replace accompanying drawing and illustrate make the copper that sputters on the glass surface and ssDNA contact after, when temperature variation, the variation of fluorescence intensity (embodiment 3).
[Figure 27] each photo represents accompanying drawing and is illustrated in the Fluirescence observation result (embodiment 4) that sputter has the onion thin skin on glass of copper.
[Figure 28] each photo represents accompanying drawing and is illustrated in the Fluirescence observation result (embodiment 4) that sputter has the human leucocyte sample on glass of copper.
[Figure 29] each photo represents accompanying drawing and is illustrated in the Fluirescence observation result (embodiment 4) that sputter has the Jurkat cell on glass of copper.
[Figure 30] each photo represents accompanying drawing and is illustrated in the Fluirescence observation result (embodiment 4) that sputter has the Jurkat cell on glass of copper.
[Figure 31] each graphic representation replaces accompanying drawing and is illustrated in to have different concns T(20) the fluorescence spectrum (embodiment 5) that obtains of oligomer DNA.
[Figure 32] each graphic representation replaces accompanying drawing and is illustrated in to have different concns T(10) the fluorescence spectrum (embodiment 5) that obtains of oligomer DNA.
[Figure 33] each graphic representation replaces accompanying drawing and is illustrated in to have different concns T(6) the fluorescence spectrum (embodiment 5) that obtains of oligomer DNA.
[Figure 34] each graphic representation replaces accompanying drawing and is illustrated in to have different concns T(5) the fluorescence spectrum (embodiment 5) that obtains of oligomer DNA.
[Figure 35] each graphic representation replaces accompanying drawing and is illustrated in to have different concns T(4) the fluorescence spectrum (embodiment 5) that obtains of oligomer DNA.
[Figure 36] each graphic representation replaces accompanying drawing and is illustrated in to have different concns T(3) the fluorescence spectrum (embodiment 5) that obtains of oligomer DNA.
[Figure 37] each graphic representation replaces accompanying drawing and is illustrated in to have different concns T(2) the fluorescence spectrum (embodiment 5) that obtains of oligomer DNA.
[Figure 38] each graphic representation replaces accompanying drawing and is illustrated in the fluorescence spectrum (embodiment 5) of the oligomer DNA acquisition that comprises different thymine alkali bases numbers.
[Figure 39] each graphic representation replace accompanying drawing and the concentration of the oligomer DNA that comprises different thymine alkali bases numbers is shown and the maximum of fluorescence intensity between mutual relationship (embodiment 5).
[Figure 40] each graphic representation replaces accompanying drawing and is illustrated in the fluorescence spectrum (embodiment 6) of the oligomer DNA acquisition with the base sequence that comprises T and C.
[Figure 41] each graphic representation replaces accompanying drawing and is illustrated in the fluorescence spectrum (embodiment 6) of the oligomer DNA acquisition with the base sequence that comprises T and C.
[Figure 42] graphic representation replaces accompanying drawing and is illustrated in to have T(10) oligomer DNA and have T(10 with the quencher modification) the fluorescence spectrum (embodiment 7) of oligomer DNA.
[Figure 43] each graphic representation replaces accompanying drawing and is illustrated in to have T(10) and the U(9) fluorescence spectrum (embodiment 8) that obtains of the oligomer DNA of G.
[Figure 44] graphic representation replaces accompanying drawing and is illustrated in to have T(10), C(10) and C(4) MeC(6) the fluorescence spectrum (embodiment 8) that obtains of oligomer DNA.
[Figure 45] graphic representation replaces accompanying drawing and is illustrated in to have T(10), A(10) and the I(9) fluorescence spectrum (embodiment 8) that obtains of the oligomer DNA of G.
Embodiment
Hereinafter, with reference to the accompanying drawings the embodiment according to present technique is described.Being for the purpose of illustration only property of embodiment described below explanation, and the exemplary embodiment of present technique is only described, and should not be construed as the scope of restriction present technique.Embodiment will be described in the following order.
＜A〉detection method of nucleic acid
1, contact procedure
(1) copper (Cu)
(2) sample
(3) reaction soln
(4) contact conditions
2, detect step
(1) irradiate light
(2) fluoroscopic examination
(3) detection of fluorescence spectrum
(4) detection of fluorescence spatial distribution
3, base sequence analysis
4, use
(1) difference of the fine gene order of detection
(2) the analyzing DNA molecule methylates
(3) nuclear observation and measurement
(4) subparticle analysis
(5) be applied to chip lab
＜B〉high-visibility pigment
＜A〉detection method of nucleic acid
Inventor's latest find the mixture emitting fluorescence of nucleic acid (DNA or RNA) and copper, as describing in detail in following examples.Find that also fluorescence spectrum and fluorescence intensity depend on the base sequence of nucleic acid and length and change, and fluorescence spectrum and fluorescence intensity depend on that whether mispairing exists and change in double-strandednucleic acid.Present technique new discovery and obtaining thus.As mentioned above, be recognized that in the past that the absorption spectrum of DNA changed by the interaction with copper, and the base sequence of DNA is depended in the change of absorption spectrum.Yet, do not know the mixture emitting fluorescence of nucleic acid and copper in the past.Fluorescence by the mixture emission has the wavelength characteristic similar to the above-mentioned fluorescence by the Cu-MT emission, but be to adopt the synthesis of oligonucleotides Nucleotide of purifying, not observed in the reaction system of containing metal thioneine (metallothionein), this is diverse with the fluorescence of being launched by Cu-MT.Hereinafter, will be to being specifically described according to the nucleic acid detection method of present technique, the fluorescent substance that its application reaches according to present technique.
Nucleic acid detection method according to present technique may further comprise the steps: the sample that contains nucleic acid is contacted with copper, and detect the fluorescence by the sample emission.In the nucleic acid detection method according to present technique, according to purpose, based on detecting detected fluorescence intensity and/or spectrum in the step, but the base sequence of analysis of nucleic acids, and can analyze mispairing in the two strands that forms nucleic acid.
1, contact procedure
In contact procedure, the sample that will contain nucleic acid contacts with copper.
(1) copper (Cu)
The form of the copper that uses in this step is the solution of cupric or the solid matter of cupric preferably.Requiring preferably to use solid matter under the situation easy and simple to handle.During nucleic acid in detection is placed on micro-scale stream in the microchip, the use of solid matter can allow copper incorporate in the microchip, thereby forms the chip structure of simplifying better.And, to compare with solution, solid matter has stable shape or performances such as vibrations, impact, heat, light, times.On the other hand, when needs shortened the reaction times, solution may be more suitable for.The form of copper can be selected rightly according to purpose.
When using copper solutions, solution preferably comprises copper (I) ion of capacity.Usually, the copper positively charged ion of divalence is stable, and the copper positively charged ion of monovalence is not as the cupric cationic stabilized.Based on this reason, preferably can make copper (II) ion be reduced to the reductive agent of copper (I) ion with CuSO 4Solution is that the cationic solution of cupric that contains of example mixes.Sodium ascorbate can be used as reductive agent.
For supply capacity copper (I) ion other method in the solution is with radiation exposure cupric (II) ion solution, to produce copper (I) ion (with reference to non-patent document 30).Perhaps, be desirably under the oxygen free condition, for example CuI, CuOTfC 6H 6[Cu (NCCH 3) 4] [PF 6] salt be dissolved in copper (I) solion (with reference to non-patent document 31) be provided in the solution of the Equivalent (2,6-lutidine, triethylamine, diisopropylethylamine, pyridine etc.) that contains acetonitrile and nitrogenous base (nitrogen base).
When using solid-state copper (solid copper), except fine copper, also can use copper-bearing alloy.The limiting examples of the shape of copper comprises powder, fine particle, rod, silk, plate and paper tinsel.Can also form the film of cupric on the surface (internal surface) of substrate or the container of the microchip that will introduce sample.
Preferably solid copper has definite shape and is placed as and makes that detected light is not blocked in detection step as described later, is not reflected etc.For example, solid copper can be placed in substrate or the container or the specific region.Solid copper can enough thinly be used up with the detection that sees through capacity.Can be configured to solid copper and sample position contacting and separate with the position of measurement from the fluorescence of sample emission, and the sample transporter that sample can be transported between the two positions is provided.At this, measurement refers to the position of the fluorescence of measure sample emission by using the irradiate light sample from the position of the fluorescence of sample emission.
The amount of the copper that contacts with sample is not particularly limited, as long as can be from the sample detection to fluorescence in detecting step.When using solid copper, the suitably setting according to the concentration of institute's cupric in sample and the area of solid matter contact, the ratio of the relative sample volume of this area, the shape that keeps the container of sample, the solid matter, the type of pollutent except copper or amount etc. of the amount of the solid matter of cupric.For example, during the copper powder that in using embodiment, uses, the preferred every ml sample 37.5mg of the amount of copper powder or more.Equally, for example, when the solid copper film forms on the surface of substrate or container (internal surface), and sample be maintained at be clipped between two sheet glass, when the degree of depth is approximately in 20 microns the space (referring to embodiment), be copper more than the 20nm at spatial surface sputter thickness at least.
(2) sample
As the sample that contains nucleic acid, can use any sample that can contain such as the nucleic acid of DNA and RNA.For example, can be nucleic acid extraction liquid, contain the solution of nucleic acid compound, such as the reaction product of the nucleic acid amplification reaction of PCR, electrophoresis sample etc.In addition, nucleic acid solution sample just also has cell itself, comprises that the tissue slice etc. of cell can use as sample.
(3) reaction soln
Sample preferably contacts in the reaction soln of saliferous with copper.Only otherwise the advantage of forfeiture present technique, the type of salt has no particular limits, and can freely select the known salt of use.For example, can freely select to use sodium-chlor (NaCl), Repone K (KCl), magnesium chloride (MgCl 2) one or more salt (referring to embodiment) in waiting.
The concentration of salt has no particular limits, only otherwise the advantage of forfeiture present technique can freely set.Preferably, the concentration of salt is set at 0.025M or higher (referring to embodiment).
Preferably, reaction soln does not contain the sequestrant compositions such as (for example EDTA, Tris etc.) that makes copper (II) ion stabilized.
(4) contact conditions
Have no particular limits the duration of contact of sample and copper, can be depending on the form of employed sample or copper and freely set.For example, when using liquid sample and copper powder, they are stirred fully, thereby can reduce duration of contact.When use has the substrate of film of solid matter of cupric or container, improve the contact area of sample and copper by the structure that changes substrate or container, thus minimizing duration of contact.In addition, when sample contacts with copper, reaction soln and contain the contact area of oxygen air and preferably be defined as duration of contact as far as possible little or short.
2, detect step
In detecting step, detect the fluorescence of the sample emission after contact procedure.
(1) rayed
At the sample that contains nucleic acid with after copper contacts, as long as can detect from the fluorescence of sample emission, for having no particular limits in order to detect the light (exciting light) that shines sample from the fluorescence of sample.
For example, mercury lamp, halogen lamp, xenon lamp, laser, LED, daylight etc. can be used as the light source use of exciting light.Can also between light source and sample, place wavelength selection system, be used for selecting desired wavelength.In this case, wavelength selection system comprises spectral filter, prism, grating mirror etc.Perhaps, as long as can be detected from the fluorescence of sample emission, from the energy transfer available light irradiation replacement such as FRET and BRET of adjacent molecule.
For effectively produce fluorescence from sample, exciting light comprises preferably having the light that is about 300 μ m～420 mum wavelengths, more preferably has the light that is about 330 μ m～380 mum wavelengths.In addition, in exciting light, the light intensity that optimal wavelength is about 420 μ m is enough low, and more preferably wavelength to be about the above light intensity of 500nm enough low so that it does not hinder the detection of fluorescence.
Preferably, excite light intensity to want enough high to detect from the fluorescence of sample emission.Preferred detection suitably arranges according to following factor with light intensity: the wavelength region of irradiates light; The size of the nucleic acid that detects, base sequence, higher structure (higher order structure), quantity and concentration; The number of signals of obtaining; The light wavelength scope that detects; The sensitivity of detector, type and structure etc.For adjusting excites light intensity, the structure of beam condensing units such as the light intensity that can suitably regulate light source type for example, send from light source, prism, type and the structure of wavelength selection system, comprise that the irradiates light of ND wave filter and aperture isocandela degree setting device is with the structure of optical system, density, range of exposures and the irradiation time that irradiation is used up.
Light such as optical fiber and mirror transmission device can be placed between the light source and sample of exciting light.The container of the maintenance sample that uses in light irradiation process has no particular limits, but preferably has material and the structure of the fluorescence that can see through the irradiates light of capacity and will detect.
(2) detection of fluorescence
The fluorescence of launching from sample can be detected by any nonrestrictive device, for example passes by known means.As detection means, can use for example photodetector, photorectifier, photomultiplier cell, ccd video camera and cmos camera etc. to be used for optical signal is converted to the element of electrical signal.Perhaps, as detection means, can adopt to take to be film or observation with the naked eye.Can shift by the energy that the fluorescence molecule adjacent with sample is brought out FRET etc., and receive from the fluorescence of fluorescence molecule emission and detect indirectly from the fluorescence of sample emission.
For the fluorescence of efficient detection from the sample emission, preferably light collecting devices such as prism are placed between sample and the detection means.For example light transmitting device such as optical fiber and mirror can be placed between sample and the detection means.
Can with rayed be in the same side or not the sample of homonymy detect fluorescence.Especially, when when detecting fluorescence with light-struck the same side, place light reflecting devices such as minute surface with different directions, thereby improve from the collection effciency of the fluorescence of sample emission.Even when when light-struck not homonymy detects fluorescence, also can place the light reflecting device with the structure that does not hinder the light detection, maybe can place for example following light reflecting device: can see through the light with illumination wavelength and reflect dichroic mirror light, that have wavelength selectivity that will detect.
When the fluorescence that detects from the sample emission, the light outside the light that detect may be arranged, comprise the scattering of light light that shines sample, from sample or keep the autofluorescence of container of sample and the light leak that comes from the outside.In this case, preferably between sample and light detection means, place the light selecting arrangement, so that the light outside the light that will detect can not arrive detection means.
The example of light selecting arrangement comprises spectral filter, prism, grating mirror etc.And, the zone of light transmission in the time of can anticipating the outside surface of the substrate of introducing sample and container or the fluoroscopic examination in the internal surface, thus only see through the light of expectation wavelength, and to rayed without any influence.
Based on the result among the embodiment as mentioned below, the preferred light selecting arrangement can only detect wavelength and be about the above fluorescence of 420nm, more preferably only detects wavelength and is about the above fluorescence of 500nm.In addition, for the influence of autofluorescence being reduced to minimum as far as possible, if necessary, only detect wavelength and be about the above light of 600nm.In the detection nucleic acid method according to present technique, for the ultraviolet excitation wavelength of about 360nm, provide the long relatively wavelength with about 600nm centre wavelength etc. and the fluorescence with long stroke skew (stroke shift).Therefore, the nucleic acid detection method according to present technique has the autofluorescence of scattered light or the emission of other materials to the less advantage of detection influence of target fluorescence.
As another light selection approach, different performances suitably is set in the time that rayed detects fluorescence afterwards with molecule is different to utilize fluorescence lifetime, gets rid of the light outside the light that will detect as far as possible, and detects essential fluorescence.
(3) fluorescence spectrum detects
Employing is applicable to the device of spectral measurement in rayed and fluoroscopic examination, measure from the fluorescence spectrum (excitation spectrum or emmission spectrum) of sample emission.
Utilize as light selecting arrangements such as spectral filter, prism, grating mirrors, by spatially or the time go up and change space or the time variation of light wavelength to measure the fluorescence intensity that will detect that shines sample, can measure excitation spectrum.By spatially or the time go up to change the light wavelength that shines sample, and by light is incorporated in the detection means, change with space or the time of measuring the fluorescence intensity that will detect, can measure emission (fluorescence) spectrum.By with the two combination, can measured excitation spectrum and emmission spectrum.
Be used for spatially changing the light that shines sample or have prism and grating mirror etc. to change the optics of optical propagation direction according to wavelength from the specific examples of the wavelength selection system of the wavelength of the fluorescence of sample emission.
Be used for changing in time the light that shines sample or comprise from the specific examples of the wavelength selection system of the wavelength of the fluorescence of sample emission: changes dissimilar spectral filters, and by control spectral filter change through light wavelength through light wavelength.Perhaps, prism and grating mirror etc. can be placed according to the optics of wavelength shift optical propagation direction, and the direction of light selecting arrangement along the specific direction propagation in the light of optics can be only selected to see through.The position of optics and/or direction of light selecting arrangement or structure can be controlled and change in time.These devices can be automatically controlled, with the light wavelength as using computer to select automatically of Change Example in time.The direction of light selecting arrangement can be by will suitably making up as the optics of slit, lens, mirror and optical fiber to dispose.
Utilize wavelength selection system, by the last excitation spectrum of light wavelength and/or the detection method of emmission spectrum of changing of time, preferably include: the light wavelength of using computer control to be selected by wavelength selection system; Utilize the device that optical signal can be converted to electrical signal of photo-detector etc. as light detection means, measuring result is read in computer; And the fluorescence intensity that records the irradiation light wavelength interrelatedly and measure.
Utilizing wavelength selection system to detect in the method for excitation spectrum and/or emmission spectrum by space change light wavelength, as light detection means, the element that receives light is arranged with one-dimensional array, and the element (as CCD and CMOS) that maybe will receive light is arranged in one plane.
(4) detection of fluorescence spatial distribution
For obtaining about the spatial distribution of nucleic acid contained in the sample and the information of shape, provide a kind of mode of spatial information to be once, the zone that nucleic acid is diffused into is to a certain degree carried out irradiation and the fluoroscopic examination of light simultaneously.Perhaps, the another kind of mode that spatial information is provided can be the site that changes detection in time, and scans the inside in the zone that nucleic acid spreads to a certain extent successively.
In the previous case, preferably use the whole surveyed area of rayed, and the light intensity that more preferably shines whole zone is uniform.Make light detection means, can utilize film or visual inspection.And, can use the light detection means that is used for optical signal is converted into the light receiving element of electrical signal that comprises two-dimensional arrangement, for example ccd video camera and cmos camera.
An example of latter event is to use laser to shine, utilize current mirror etc. to change the irradiation position of laser in time, detection is from the fluorescence of the irradiation position of laser emission, and from the data that the irradiation position of laser is related with detected fluorescence intensity the spatial distribution of acquisition fluorescence.In this case, preferably by utilizing current mirror control rayed position, make up by computer recording rayed position and in computer the data of detected fluorescence intensity with at that time rayed location association, thus the spatial distribution of analysis of fluorescence intensity automatically.
Acquisition is that the one dimension that the light detection means that adopts light-struck light source linearly and comprise the light receiving element (as linear transducer) that one dimension is placed is measured fluorescence distributes about the another way of the information of spatial distribution, and mobile its position sequentially.Perhaps, by using such as the light receiving element of the spatial distribution that can obtain fluorescence intensity once of ccd video camera and cmos camera and the area of space that movement sequentially will be observed, can obtain the spatial distribution of the fluorescence intensity of broader area.In these cases, also expectation is suitably used computer to control surveyed area and is recorded the fluoroscopic examination result, and based on the spatial distribution of the automatic analysis of fluorescence intensity of these information.
3, base sequence analysis
Next, with the method for description based on the mispairing in the two strands of the base sequence of the information analysis nucleic acid of the fluorescence that in detecting step, detects and formation nucleic acid.
The spatial distribution and the time that comprise fluorescence intensity and/or spectrum (excitation spectrum or emmission spectrum), fluorescence lifetime, fluorescence about the object lesson of the information of fluorescence change.These information can be converted into numeral, the record on computers or computing on computers for analysis.
The fluorescence intensity that obtains in detection method depends on concentration, type, size, higher structure and the base sequence (referring to embodiment) of reaction conditions, optical system and nucleic acid.Especially, when type, size, higher structure and the base sequence of reaction conditions, whole optical system, nucleic acid is constant, can provide by the measuring result of fluorescence intensity about the information of the concentration of nucleic acid.In this case, analytical procedure may further comprise the steps: information about the concentration known of the nucleic acid that will detect and the relation between the fluorescence intensity is provided, measure expectation two or more concentration nucleic acid fluorescence intensity, set up calibration curve, will be applied to by the fluorescence intensity that detection provides in the relation between concentration and the intensity, and calculate the concentration of nucleic acid.This method also can be by recording the relation between nucleic acid concentration and the fluorescence intensity in advance, and calculate nucleic acid concentration and automatically perform from fluorescence intensity on computers.
In addition, especially, as long as concentration, reaction conditions and higher structure are constant, base sequence and higher structure (referring to embodiment) that the spectrum that obtains in detecting step and fluorescence intensity depend on nucleic acid." higher structure " refers to single-stranded structure or the duplex structure of nucleic acid here, and relates to that the two strands that is caused by hybridization forms or unparalleled chain formation and site thereof, and the mispairing in two strands or do not have mispairing and the site.These characteristics can be used for test result by fluorescence spectrum and intensity provides information about base sequence and the higher structure of nucleic acid.More specifically, when the sequence of the nucleic acid that comprises in the known sample and higher structure are among the known a limited number of candidate any one, can be by fluorescence spectrum and the intensity of measuring each candidate in advance, and estimate sequence and the higher structure of the nucleic acid that comprises in the sample of fluoroscopic examination with the measuring result comparison.The method of more two or more spectrum comprises computation of characteristic values, the ratio of maximum excitation wavelength and fluorescence intensity, the fluorescence intensity in two or more wavelength region may for example, and relatively they.In addition, can calculate for the difference between two spectrum relatively, to quantize similarity.Perhaps, when only when paying close attention to its shape and come comparison spectrum, can be that identical value (uniform value) compares by the maximum strength of proofreading and correct institute's photometry spectrum.In addition, fluorescence intensity multiply by variable and utilizes method of least squares, and the difference between two spectrum is that the variate-value at minimum place and the big I of the difference between two spectrum at this point are quantized.Sequence by recording in advance nucleic acid on computers and the relation between higher structure and spectrum and the fluorescence intensity, and estimate sequence and the higher structure of nucleic acid on computers from the information of measured spectrum and fluorescence intensity, these operations can be carried out automatically.
When the spatial distribution that detecting the fluorescence that obtains in the step and time being changed when analyzing, this this space can be by visual control, the feature of observing this space and qualitative classification.Perhaps, fluoroscopic image can be imported in the computer, and can handle by image and carry out quantitative analysis.One of example of handling as image, for example extract the zone of emitting fluorescence by binary processing, and can calculate numerical value, as fluorescence intensity at this regional area, skeletal lines length, circularity, center or position of centre of gravity, summation, mean value, average, intermediate value, dispersiveness and standard deviation.Perhaps, pattern match (pattern matching), learning algorithm etc. can be applicable to have specified shape the zone identification or be applied to shape and sort out.
4, use
By above-mentioned contact procedure and fluorescence step are combined with the base sequence analysis where necessary, can be applicable in the various fields according to the nucleic acid detection method of present technique.Hereinafter, with the application of describing according to the nucleic acid detection method of present technique.
(1) detects the difference of fine gene order
According to present technique, by measuring fluorescence spectrum and/or intensity, can provide the information about the base sequence of nucleic acid contained in the sample.Yet when sample comprised multiple nucleic acid, detected fluorescence spectrum and/or intensity quilt were average, the possibility that has the difference between the fine gene order not to be identified.Therefore, where necessary, be preferably as follows the described several different methods that will limit the scope of the nucleic acid that will detect of literary composition and make up.
The embodiment of the difference between the fine gene order comprises single nucleotide polymorphism (SNPs).The base sequence of known analysis of nucleic acids is very useful for medical diagnosis on disease.As everyone knows, by analyzing the risk that SNPs can assess the various diseases that comprises heart trouble.
First example of a kind of method that limits the scope of nucleic acid to be detected comprises the method for only taking out the part that contains sequence to be analyzed in the nucleic acid contained from sample.Particularly, this method comprises the PCR that adopts hybridization, electrophoresis, the employing nucleic acid amplification technologies be fixed on the dna probe on substrate or the pearl and carry out to be used for amplifying sequence to be analyzed etc.In addition, restriction enzyme reaction and ligation can combine with these methods.
Second example only comprises from sample the method for selectivity picked up signal in the particular bases of contained nucleic acid.Particularly, utilize the phenomenon of in present technique, finding, namely in the thymus pyrimidine that does not contain complementary strand (T) sequence, detected very strong fluorescence.In this method, prepared sequence hybridization that the site with sudden change to be detected in the contained nucleic acid of sample is excluded in advance to form one or more probe nucleic acids of double-strandednucleic acid.Probe nucleic acid with to detect the nucleic acid hybridization of the sudden change in the gene order with sampling.In probe nucleic acid, when producing sudden change, when for example the VITAMIN B4 that will analyze in the sequence (A) is replaced by other bases, T will be placed in the sequence of the probe corresponding with this site.Like this, the base when this site is replaced by any base beyond the A, and the mispairing of T namely produces, and measures strong fluorescence thus.Perhaps, when producing sudden change, when for example the T that will analyze in the sequence is replaced by other bases, the base outside the A will be placed in the sequence of the probe corresponding with this position.Like this, when not suddenling change, never form double-stranded T place and measure strong fluorescence.
Probe nucleic acid can be by DNA, RNA, peptide nucleic acid(PNA) (PNA), the low polynucleic acid of thiophosphatephosphorothioate (phosphorothioate) type, BNA(LNA) etc. formation.
The 3rd example is included in to detect to limit in the step wants irradiates light maybe will detect the method for the physical region of fluorescence.For example, the light that dies near field of light such as wink is used to shine sample, and namely light only is radiated at the specific region of special restriction.This method can combine with the means (means, device) of the nucleic acid that keeps at specific position or move.By the means at specific position maintenance or mobile nucleic acid, nucleic acid is fixed on solid surface, by the very fine stream as nanoporous, or mobile in protein such as enzyme.
Energy shifts, and as FRET and BRET, can be used as restriction and wants irradiation light maybe will detect the additive method of the physical region of fluorescence.Under rayed, the molecule that is used for bringing out FRET and BRET is arranged in and adjoins zone to be detected, and the energy of this molecule shifts and is used to the local light irradiation certainly.In fluoroscopic examination, be used for being arranged in and adjoining zone to be detected by absorb fluorescence molecule that fluorescent energy brings out FRET from sample, detect the fluorescence that from fluorescence molecule, produces to be used for local fluoroscopic examination.
(2) methylation analysis of dna molecular
Known cytosine(Cyt) (C) in dna molecular is methylated in intracellular genome.Whether whether methylating of cytosine(Cyt) (C) exist and can be found by uridylic (U) replacement by judging cytosine(Cyt) (C).In other words, when handling nucleic acid under proper condition with hydrosulfate, have only unmethylated cytosine(Cyt) (C) can optionally be converted into uridylic (U).Therefore, when detecting uridylic (U), can detect the existence of the non-cytosine(Cyt) that methylates.
In uridylic and thymus pyrimidine, very strong from the fluorescence of the mixture of nucleic acid and copper, but in cytosine(Cyt) and the cytosine(Cyt) that methylates, detect less than (with reference to embodiment).Therefore, handle by hydrosulfate, the non-cytosine(Cyt) that methylates that comprises in the sample optionally is converted into uridylic, and the variable quantity of detected fluorescence intensity and/or spectrum in definite sample, analyze thus in nucleic acid cytosine(Cyt) methylate or the existence of demethylation whether with and amount.The method of explanation in above-mentioned by being used in combination " difference of fine gene order detects ", the position of methylate in the base sequence of nucleic acid cytosine(Cyt) or demethylation cytosine(Cyt) etc. can at large be analyzed.
Methylation analysis can carry out according to the following steps: at first, according to technique known in the past, handle the sample that contains nucleic acid with hydrosulfate.Then, sample detection intensity of fluorescence and/or spectrum after the sample before handling from hydrosulfate and hydrosulfate are handled.Afterwards, detected intensity of fluorescence and/or spectrum the sample after relatively the sample before hydrosulfate is handled and hydrosulfate are handled.The amount of the non-cytosine(Cyt) that methylates is more many, and the amount of the uridylic that is produced by the hydrosulfate processing is just more many.By the fluorescence of the sample before and after relatively hydrosulfate is handled, can provide methylating or information that whether demethylation exists and measure about the cytosine(Cyt) in nucleic acid.
When comprising a large amount of thymus pyrimidines in the base sequence of the nucleic acid that comprises in the sample, the fluorescence of thymus pyrimidine just becomes noise, may reduce from the signal/noise ratio of the fluorescence of uridylic.In addition, handle the base sequence that a plurality of non-cytosine(Cyt) that methylates that is converted into uridylic may be present in nucleic acid by hydrosulfate.In this case, combine by following method, can effectively analyze methylating at the base sequence of the nucleic acid of specific region.
At first, with the nucleic acid that comprises in the sample after hydrosulfate is handled in the zone amplification of wanting analysis of methylation or concentrate.Particularly, use nucleic acid amplification such as PCR to send out and utilize the nucleic acid method of enrichment of nucleic acid hybridization reaction.
Next, fluorescence that inhibition produces from other zones outside the c zone of wanting analysis of methylation.In the thymus pyrimidine of fluorescence in single stranded DNA from the mixture of nucleic acid and copper very high intensity is arranged, and significantly suppressed (with reference to embodiment) in the thymus pyrimidine in double-stranded DNA.Therefore, will have analysis and hybridize with nucleic acid chains with the mask (mask) of the complementary base sequence of exterior domain, suppress the fluorescence from the thymus pyrimidine in the zone beyond analyzing, can effectively detect the fluorescence from zone to be analyzed thus.Perhaps, quencher can be used for suppressing from analyzing the fluorescence that zone in addition produces.By quencher being positioned over the position of contiguous mixture, can be suppressed (with reference to embodiment) from the fluorescence of the mixture of nucleic acid and copper.Therefore, by quencher being placed on the zone beyond analyzing, can be with the fluorescence of high-level efficiency detection from zone to be analyzed.
The 3rd, have only methylated zone to be analyzed optionally to be excited, or have only regional since then fluorescence optionally to be detected.Particularly, the mixture that can excite nucleic acid and copper is arranged in adjacent with zone to be analyzed with the alms giver's probe that produces fluorescence, and the energy by FRET, BRET etc. shifts and is used to only selective excitation zone to be analyzed.Like this, only the fluorescence from zone to be analyzed can be detected.Perhaps, shifting the acceptor probe that excites and launch the fluorescence with different wave length by the energy from the fluorescence of the mixture of nucleic acid and copper can be arranged in adjacent with zone to be analyzed, and by detecting the fluorescence that is sent by acceptor probe, can detect the fluorescence from zone to be analyzed.Aforesaid method can be with any Combination application.
(3) nuclear observation and measurement
According to present technique, the detection method of nucleic acid can be applicable to include the sample of karyocyte, to detect the spatial distribution of nucleic acid.Analyze its distribution and shape to provide about information such as the nuclear distribution in tissue slice or cell, position, quantity, size, shapes.
Can go out the quantity of karyocyte from these information calculations.The interior shape of the container that sample is introduced into is suitably designed, to keep the sample of constant volume, the concentration of the karyocyte that this can be used for comprising in the measure sample in constant area.In addition, when measuring nuclear shape and quantity simultaneously, can identify or count the cell that comprises difform nuclear a plurality of types.For example, the with good grounds particulate state white corpuscle of known white corpuscle, monocyte are with lymphocyte and different nuclear shapes.Nucleic acid detection method according to present technique can be used for identification or counts leukocytic kind.
As the parasitic known plasmodial type that causes malaria, known pernicious malaria protozoon, Plasmodium vivax, communicable subtropical diseases plasmodium, Plasmodium ovale etc.In addition, as the stage, known ring bodies (ring form), nourishing body, schizont, gamont.In order suitably to design the treatment plan of infected patient, it is very important suitably identifying these types.Although the short-cut method that detects gene or antigen is arranged, common in order to identify plasmodial type, the shape that dyes to observe nuclear by Ji's nurse Sa in principle.Yet in this method, the problem of existence is when dyeing is insufficient, may cause false diagnosis.In contrast, in case be applied to the identification of plasmodium type according to the nucleic acid detection method of present technique, no longer need the normal dyeing agent.Therefore, easier, the reliable method diagnosis that can need not clean by simple dyeing such as malaria.
(4) analysis of subparticle
When detection comprised or be fixed on nucleic acid in the subparticle (for example being included in cell or pearl in the liquid sample), particle can be static, or flowed in microfluidic circuit.For example, liquid sample is introduced in the flow cell (flow cell) together with sheath stream, and be clipped in the middle to form laminar flow by sheath stream.The fluorescence that is produced by the particle that flows through flow cell can be detected.Flow cell can have arbitrarily be widely studied as low cytometric analysis, the structure of exploitation and practical application.
(5) application in the chip lab
Nucleic acid detection method according to present technique can be incorporated into chip lab, for the treatment of or detect sample in the container such as microfluidic circuit chip.In this case, according to application target, for more convenient, the pretreatment sample step is introduced into and is combined in the container.
The sample pretreatment step that is used for the detection of nucleic acid and sequential analysis for example comprises extracts, separates and amplification of nucleic acid.Example comprises the separation that utilizes electrophoresis, gel-filtration column or absorption column, the amplification by the PCR reaction etc. more specifically.These means can be attached in the microchip by known technology.
Perhaps, as being used for the sample pretreatment step that observation, detection and analysis package are contained in the nuclear of cell, can select or concentrated specific cells.As the different qualities of selecting or the step of concentrated specific cells, can utilize to depend on cell category (for example size, proportion, toughness, to the bonding force of predetermined substance such as antibody etc.As an example, to the cell that will observe or do not want observed cell and have the antibody of specificity combination to be fixed on the internal surface or pearl of container.By combination or the debond that utilizes antagonist, can select or concentrating cells.In addition, preparation fixedly has the subparticle of the magnetic substance of antibody.Utilize magnetic, can only select the cell that is adsorbed with the cell of magnetic substance or does not adsorb magnetic substance.In addition, sample is in osmotic pressure, acid or the alkali of suitable variation, whereby can be only with hematoclasis and removal, only to select white corpuscle.As another example, for example, when the plasmodium in the observation human blood, be known that in the red corpuscle that is infected by plasmodium, to form the magnetic substance that is called as " haemozoin " in the past.This red corpuscle can be separated or concentrated by magnetic.This method is combined with nucleic acid detection method according to present technique, can observe plasmodium easy, reliably.
＜B〉high-visibility pigment
Next, with the fluorescent substance that specifically describes according to present technique.
For utilize fluorescent microscope, flow cytometer, gene amplification reaction, gene sequencing reaction, quantitative assay comprise protein biomolecules, measure binding ability observation between the biomolecules comprise protein and analysis of cells, tissue, biomolecules, application has the pigment of versicolor emitting fluorescence, as fluorescein and phycoerythrin.High-visibility pigment is used as instrument, so that the information about the localization of the biomolecules of pigment institute combination to be provided, and provide about the position of the molecule (s) of interest by the identification of antibody or nucleic acid probe (for example, by pigment is combined on antibody or the nucleic acid probe) and the information of amount.When preparing to have some pigment of different colours, can analyze more molecule (s) of interest.
Application can generate the fluorescent substance with multiple spectrum according to the nucleic acid detection method technology of technology mentioned above.In other words, nucleic acid is launched the fluorescence with different spectrum or intensity with the mixture of copper according to base sequence and the length of nucleic acid.The characteristic of the mixture by utilizing nucleic acid and copper, mixture can be used as the fluorescent substance of launching multiple spectrum, for example, can be used as the high-visibility pigment of traget antibody.
Present technique can have following configuration:
(1) a kind of nucleic acid detection method that may further comprise the steps:
The sample that contains nucleic acid is contacted with copper, and
Detection is from the fluorescence of sample emission.
(2) according to the detection method of above-mentioned (1), wherein,
Come the base sequence of analysis of nucleic acids based on detected intensity of fluorescence and/or spectrum in detecting step.
(3) according to the detection method of above-mentioned (1), wherein
Based on detected intensity of fluorescence and/or spectrum in detecting step, analyze the mispairing in the two strands that forms nucleic acid.
(4) according to the detection method of above-mentioned (1), may further comprise the steps:
Handle sample with hydrosulfate, wherein
Based in detecting step, sample detection before handling from hydrosulfate to intensity of fluorescence and/or spectrum with from the sample detection after the hydrosulfate processing to intensity of fluorescence and/or the difference (difference the spectrum, difference), the cytosine(Cyt) in the analysis of nucleic acids methylates.
(5) according to the arbitrary detection method in above-mentioned (1) to (4), wherein copper is solid copper.
(6) according to the arbitrary detection method in above-mentioned (1) to (5), wherein contact procedure is under the coexistence of salt, the step that sample is contacted with copper.
(7) according to the arbitrary detection method in above-mentioned (1) to (6), wherein
Detect step and be and detect by with wavelength being the rayed sample of 300 μ m to 420 μ m and the step of the fluorescence from sample, launched.
Embodiment 1
Embodiment 1 illustrate when nucleic acid with contain reduce Cu(II with xitix) ion and the Cu(I that produces) after ion solution mixes, under given conditions, launch orange fluorescence by UV-irradiation.
＜material and method 〉
Cu:CuSO 4Solution and (+)-L-sodium ascorbate (abbreviating " S.A. " hereinafter as) are bought from Sigma-Aldrich company.
Nucleic acid: use from BioDynamics laboratory Inc.(Tokyo, Japan) salmon sperm dna (Sonicated Salmon Sperm DNA) (abbreviating " ssDNA " hereinafter as) of the supersound process of buying.In addition, the Custom Oligo that buys from Invitrogen Corporation is used as oligomeric-DNA.
Damping fluid: from DOJINDO Laboratories(Kumamoto, Japan) HEPPSO of Gou Maiing regulates the use of pH to 8.5 back according to the guide that is provided by manufacturer.
Fluorophotometer: use NanoDrop3300(Thermo Fisher Scientific, Inc., Waltham, MA, USA) or F-4500 type spectrophotofluorometer (Hitachi High-Technologies Corporation).In NanoDrop3300, use the ultraviolet LED light source that exciting light is provided.Measurement is by the fluorescence spectrum of excitation.Adopt software kit, obtained the relative fluorescence units (RFU) that fluorescence intensity becomes peaked wavelength correspondence, as peak value RFU value.In F-4500 type spectrophotofluorometer, used the Accessories by Helix Biomedical, quartz capillary and special-purpose aptamers cell (dedicated adapter cell) that Inc. produces.Unless explanation is hereinafter arranged in addition, otherwise that use is NanoDrop3300.
Spectrophotometer: the NanoDrop1000 spectrophotometer is used to measure absorption spectrum.
The HEPPSO damping fluid of sample preparation and fluorometric assay: 50mM and sodium-chlor (250mM), CuSO 4(0mM～4mM), S.A.(4,50mM), ssDNA(1mg/ml) or oligomer DNA (50,250,500 μ M) mix so that 20 μ l samples to be provided.Known S.A. has the CuSO in the solution 4The Cu(II that produces) ion is reduced to Cu(I) effect (with reference to non-patent document 31).
＜result 〉
Fig. 1 and Fig. 2 are illustrated under the condition that S.A. concentration is 50mM separately, by changing CuSO 4Concentration and the fluorescence spectrum and the RFU value that obtain; (A) show fluorescence spectrum, (B) show peak value RFU value (peak RFU value).
Fig. 3 and Fig. 4 are illustrated in CuSO separately 4Concentration is that 0.4mM and S.A. concentration are under the condition of 4mM, the fluorescence spectrum of acquisition.Concentration with oligomer DNA of 20,10,6 and 3 base length is respectively 50 μ M, 50 μ M, 250 μ M and 500 μ M.Fig. 3 illustrates has sequence numbering (SEQ ID NO): the result of the oligomer DNA of the base sequence described in 1～6.Transverse axis is represented wavelength, and (A) longitudinal axis in is represented the RFU value of each wavelength correspondence, and (B) longitudinal axis in is represented the value that obtains except the RFU value under each wavelength with maximum RFU value.Fig. 4 shows the oligomer DNA (being described as T(20 hereinafter) that has in the sequence numbering 2 base sequence of describing) result (A), oligomer DNA (being described as T(6 hereinafter) with base sequence of describing in the sequence numbering 10) result (B), have sequence numbering: result (C) oligomer DNA (being described as T(3 hereinafter) of the base sequence of describing in 12) has the oligomer DNA (being described as T(3 hereinafter) of the base sequence of describing in the sequence numbering 11) result (D).Transverse axis is separately represented wavelength, and the longitudinal axis is separately represented the RFU value under each wavelength.
As shown in the figure, the pattern of fluorescence spectrum figure (pattern, pattern) (peak wavelength and intensity) depends on the base sequence of nucleic acid and changes.
Next be, at CuSO 4Concentration 0.4mM and S.A. concentration are under the condition of 4mM, measure at oligomer DNA T(20), T(6) and the fluorescence spectrum that T(3) obtains and absorption spectrum are over time.Be right after for the first time measure fluorescence spectrum and absorption spectrum before, add S.A..After 8,14,24 and 35 minutes, measure fluorescence spectrum and absorption spectrum.The results are shown among Fig. 5 and Fig. 6.In Fig. 5, top figure illustrates the fluorescence spectrum that the longitudinal axis is RFU value (absolute value) separately, and middle figure illustrates the fluorescence spectrum that the longitudinal axis is RFU value (relative value) separately, and following figure illustrates absorption spectrum separately.Fig. 6 illustrates peak value RFU value (A) over time, and when wavelength being shown being 346nm (B) over time.
As shown in the figure, at all oligomer DNA T(20), T(6) and T(3) in, after 30 minutes, fluorescence almost disappears.Especially, in the short oligomer DNA of base length, fluorescence rapidly disappears.After 35 minutes, the test fluorescence spectrum.After this S.A. solution that adds 1.8 μ l concentration immediately again and be 44mM in the sample to measure.Can detect fluorescence again.Thus, the disappearance of fluorescence can be thought because Cu(I) ionic oxide formation is Cu(II) ion.At oligomer DNA T(6) and T(3) in separately the fluorescence spectrum, along with reducing of peak strength, observe new peak at short wavelength side.
On the other hand, in each oligomer DNA absorption spectrum separately, observe peak strength reduction in time.Compare with fluorescence spectrum, absorption spectrum reduces more lentamente.
Fig. 7 (A) to Fig. 7 (C) illustrate by F-4500 type spectrophotofluorometer detect from oligomer DNA T(20), T(6) and the two-dimensional fluorescence spectrum that T(3) obtains.Fig. 8 is illustrated in excitation spectrum (dotted line) and the fluorescence spectrum (solid line) that each oligomer DNA obtains.This spectrogram is with the wavelength of fluorescence of 1nm at interval and the excitation wavelength interval measurement of 2nm.
As shown in the figure, the pattern that has confirmed fluorescence spectrum depends on the base length of oligomer DNA and changes.The pattern that has also confirmed excitation spectrum depends on base length and changes.
Be further to investigate the relation between base sequence and the spectrum, measured the fluorescence that has separately by the oligomer DNA of three base length sequences of sequence numbering 11 and 18 VITAMIN B4 of describing (A) and thymus pyrimidine (T) combination acquisition.The results are shown among Fig. 9 and Figure 10.In Fig. 9, the longitudinal axis (A) represents by the RFU value under each wavelength of Nanodrop test, and the longitudinal axis (B) is represented to remove the RFU value under each wavelength and the value that obtains with maximum RFU value.Figure 10 illustrates mean value and the standard error that the maximum value of RFU and peak wavelength three times are measured.
As shown in the figure, confirmed that fluorescence intensity and peak wavelength depend on the base sequence of oligomer DNA and change.
Figure 11 is illustrated in the test result of the oligomer DNA acquisition that comprises sequence numbering 19 and 20 sequences of describing.Although confirmed a little less than the fluorescence intensity in the oligomer DNA with sequence that sequence numbering 20 describes, the fluorescence with oligomer DNA emission of the sequence that the sequence numbering 20 that contains uridylic (U) describes has similar spectral shape and peak position with the fluorescence that the oligomer DNA of the sequence with sequence numbering 19 descriptions that contains thymus pyrimidine (T) is launched.
＜discuss
Present embodiment shows, when DNA with wherein be mixed with CuSO 4When mixing with the HEPPSO buffered soln of the sodium chloride-containing of S.A., observe the fluorescent orange that wavelength is approximately 500nm～700nm by UV-irradiation.Confirmed that fluorescence intensity depends on CuSO 4Concentration, and fluorescence intensity and spectrum are influenced by the base sequence of nucleic acid all.
In the oligomer DNA that comprises thymus pyrimidine (T), VITAMIN B4 (A) or uridylic (U) at least, observe fluorescence.In the test of using the oligomer DNA that respectively has three base length that comprise thymus pyrimidine (T) and VITAMIN B4 (A), in any sequence, all observe fluorescence.In addition, show that also fluorescence intensity and spectrum not only are subjected to the amount influence of thymus pyrimidine (T) or VITAMIN B4 (A), also are subjected to the influence of the position (sequence order) on oligomer DNA.
After adding S.A., prolongation in time, fluorescence intensity reduces in time, but owing to the adding again of S.A. recovers.In a single day simultaneously, Cu(I) ion is very unstable in the presence of oxygen, and has lost the reductive action of S.A., Cu(I) just change Cu(II into) or solid copper.Thus, think that fluorescence is to come from Cu(I) mixture of ion and nucleic acid.In order to detect fluorescence by the interaction between copper and the nucleic acid, expectation minimizes reaction soln with contacting of airborne oxygen.
Embodiment 2
Embodiment 2 illustrates when the solution that contains nucleic acid contacts with solid copper,, launches the fluorescent orange similar to observed fluorescence among the embodiment 1 by UV-irradiation under certain condition.
＜material and method 〉
Copper powder (copper, powder ,-75 μ m, 99.9%/Cat.No.030-18352/ are by Wako Pure Chemical Industries, and Ltd. produces, Osaka, Japan) is used as the copper that contacts with nucleic acid.
As RNA, use the total RNA(Rat Brain of rat brain Total RNA) (Cat.No.636622, Takara Bio Inc., big Tianjin, Japan), it is dissolved in the water of handling through DEPC (Cat.No.312-90201/Wako pure chemistry Industrial Co., Ltd, Japan).
PIPES, ACES, BES, TAPSO, HEPPSO, EPPS, TAPS, CAPS, TES, three (methylol) methylglycine (Tricine) and OPSO are from DOJINDO Laboratories(Kumamoto, Japan) buy.Wherein each regulated pH value back according to the guide that is provided by manufacturer and used.The same among other reagent and the embodiment 1.
Be in 40 milliliters the solution by various nucleic acid, salt and copper powder being mixed into total amount, and stir 15 minutes, thereby nucleic acid is contacted with copper.Except as otherwise noted, otherwise the add-on of copper powder is every ml soln 375mg.Except as otherwise noted, otherwise the amount of salt or sodium-chlor (NaCl) is 500mM.
So that after the copper powder sedimentation, measure fluorescence spectrum and the intensity of supernatant liquid in that sample is centrifugal.Step similar among the measurement employing of fluorescence spectrum and intensity and the embodiment 1 is implemented.
＜result 〉
Reaction soln to the ssDNA that added 1.5mg/ml is surveyed 3 fluorescence.The results are shown in (transverse axis: wavelength, the longitudinal axis: RFU) among Figure 12.As shown in the figure, contact with solid copper, when using ultraviolet excitation then, can detect at about 600nm place and have the fluorescence at peak when the sample that contains nucleic acid.
Next, prepare reaction soln by add the copper powder that quantity is respectively 375mg, 250mg, 125mg, 62.5mg, 37.5mg, 12.5mg and 0mg based on the reaction soln of 1mL.The ssDNA of 1.5mg/ml is added in the reaction soln.Survey 3 times fluorescence.The results are shown among Figure 13.As shown in the figure, fluorescence intensity depends on the amount of copper powder.In the present embodiment in the employed copper powder, when content is 37.5mg/ml or more for a long time, can be observed tangible fluorescence.On the other hand, when content is 12.5mg/ml or still less the time, do not observe tangible fluorescence.
Then, change type and the concentration of salt in the reaction soln.The ssDNA of 1.5mg/ml is joined in the reaction soln.The fluorescence intensity that relatively records.The results are shown among Figure 14.(A) be illustrated in the fluorescence intensity that records in the reaction soln that adds 0.5M, 0.25M, 0.1M, 0.05M, 0.025M and 0M sodium-chlor (NaCl).(B) be illustrated in adding 0.45M sodium-chlor (NaCl), 0.45M Repone K (KCl), 0.45M magnesium chloride (MgCl 2) and the reaction soln of 45% ethanol (EtOH) in the fluorescence intensity that records.Fluorescence intensity is represented with the RFU at 604nm place, surveys 3 times.The result illustrates with mean value and standard error.As shown in the figure, fluorescence intensity depends on the amount of sodium-chlor.In addition, under Repone K and magnesium chloride and sodium-chlor coexistence, detect fluorescence.
When Figure 15 illustrates nucleic acid concentration in joining reaction soln and changes, the comparative result of the fluorescence intensity that records.(A) be illustrated in the fluorescence intensity that records in the reaction soln of the ssDNA that adds 5mg/ml, 2.5mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml, 0.05mg/ml and 0mg/ml.(B) be illustrated in the fluorescence intensity that records in the reaction soln of the RNA that adds 2.5mg/ml, 0.25mg/ml and 0mg/ml.Transverse axis is represented the concentration of nucleic acid, and the longitudinal axis is illustrated in the RFU that wavelength of fluorescence is the 604nm place.Test is carried out 3 times.The concentration of sodium-chlor (NaCl) is 0.25M, and the amount of copper powder is 200mg/ml.Except as otherwise noted, use this condition otherwise in the experiment below.As shown in the figure, fluorescence intensity depends on the concentration of DNA and the concentration of RNA.
Next, measure and to have added 0.1mM and have sequence numbering: 1, the fluorescence of the reaction soln of 2,5, the 6 and 9 not homotactic oligomer DNAs of describing.The results are shown among Figure 16.(A) longitudinal axis of figure is represented the RFU value that recorded by Nanodrop, and (B) longitudinal axis of figure is represented the relative RFU value when peak height is set to 1.As shown in the figure, fluorescence intensity and peak wavelength are influenced by base sequence.Especially, susceptible of proof is when the per-cent of thymus pyrimidine (T) is high, and fluorescence intensity is also high, and peak wavelength becomes longer.
Also adopt F-4500 type spectrophotofluorometer to test and have sequence numbering: 1, the reaction soln of the oligomer DNA of 2,5 and 6 sequences of describing.Figure 17 illustrates when the exciting light (slit width is 10nm) with 360nm shines, the result of the fluorescence spectrum in 400nm～700nm (slit width is 2.5nm).Reconfirm that in the sequence that contains thymus pyrimidine (T) and VITAMIN B4 (A), when the per-cent of thymus pyrimidine (T) was high, fluorescence intensity was also high, and peak wavelength becomes longer.It is 3nm that Figure 18 illustrates by scanning 330nm～390nm(slit width) and 400nm～700nm(slit width be 2.5nm) the exciting light measurement excite-result of fluorescence spectrum.(A) three-dimensional plot is shown, and (B) level line is shown.The EX axle is represented excitation wavelength (nm), and the EM axle is represented wavelength of fluorescence (nm), and short transverse is represented fluorescence intensity.Based on these results, can recognize to excite with fluorescence spectrum and the intensity Different Alkali basic sequence owing to DNA to change.
Fluorescence for the oligomer DNA of the composite sequence of the thymus pyrimidine (T) of further investigating the relation between base sequence and the spectrum, measured the cytosine(Cyt) (C) that respectively has sequence numbering 21 and 26 8 bases describing and 12 bases.The results are shown among Figure 19.As shown in the figure, when sequence not simultaneously, even the based composition of DNA is identical, fluorescence intensity is also different.
Next, measured the pattern of the fluorescence spectrum of the double-stranded DNA that comprises mispairing.As double-stranded DNA, use following three types: respectively have the oligomer DNA of the sequence shown in the sequence numbering 1 and the mixture ((e)+(f)) that respectively has the oligomer DNA of the sequence shown in the sequence numbering 2, respectively have the oligomer DNA of the sequence shown in the sequence numbering 5 and the mixture ((d)+(f)) that respectively has the oligomer DNA of the sequence shown in the sequence numbering 2, respectively have the oligomer DNA of the sequence shown in the sequence numbering 1 and the mixture ((e)+(c)) that respectively has the oligomer DNA of the sequence shown in the sequence numbering 6.Any oligomer DNA all mixes with the ultimate density of 0.5mg/ml.The results are shown among Figure 20.(A) the longitudinal axis is represented the RFU value that recorded by Nanodrop, and the longitudinal axis (B) is represented the relative RFU value when peak height is set to 1.Transverse axis is represented wavelength (nm).As shown in the figure, low in the fluorescence intensity ratio single stranded DNA in the double-stranded DNA.Yet, in the double-stranded DNA of the mispairing with thymus pyrimidine (T), confirm to have strong fluorescence.
Compared the fluorescence intensity that when the pH value in buffer type and the reaction soln changes, records.The results are shown among Figure 21.(A) relative value of the sample (+) that contains ssDNA and the sample (-) that does not the contain nucleic acid peak value RFU value under each buffer condition is shown.(B) illustrate under the same conditions, contain the relative value of the peak value RFU value of the sample with oligomer DNA that the sequence shown in the sequence numbering 1 is arranged.(C) illustrate under the same conditions, contain the relative value of peak value RFU value of the sample of the oligomer DNA with the sequence shown in the sequence numbering 2.The concentration of each damping fluid is 50mM, and the ultimate density of ssDNA is 0.5mg/ml, and the ultimate density of oligomer DNA is 25mM.The relative value of peak value RFU value refers to that the peak value RFU value that will record is set at 1 under non-cushioned condition.Fluorescence intensity depends on the type of damping fluid.When free nucleic acid exists in damping fluid, almost can not detect fluorescence.
＜discuss
Based on the result in the present embodiment, show when nucleic acid contacts with the solid copper powder, comprising under the felicity condition of salt concentration, with nucleic acid and Cu(I) ion situation about contacting is the same, can detect fluorescence.As if observed fluorescence is to provide with identical mechanism in the various situations of cupric ion and solid copper, because their characteristic as wavelength characteristic and sequence dependent, almost is identical.In addition, when RNA is used as nucleic acid, also observe fluorescence.In addition, in double-stranded DNA, when especially in thymus pyrimidine (T), having mispairing, observe strong fluorescence.This shows, may suppress to be combined the generation of the fluorescent substance that causes with copper by nucleic acid with the combination of complementary sequence.In addition, think that increase in the fluorescence intensity in mispairing site can be applicable to detect the method for the variation in the base sequence at nucleic acid.
In the experiment that is used for the fluorescence under the more various buffer conditions, in the damping fluid of PIPES, BES, HEPPSO, EPPS, TAPS, CAPS, TES and POPSO, observe fluorescence.Especially, in the damping fluid of PIPES, HEPPSO, EPPS and POPSO, detect strong fluorescence.In the pH scope between 7.0～10.5, can be observed fluorescence.Discovery depends on that the variation of the fluorescence intensity of buffer type and pH value presents the different pattern of the base sequence that depends on nucleic acid.On the other hand, have chelating and stablize Cu(II) damping fluid of ion characteristic has the tendency that does not observe fluorescence.Although data are not provided in the present embodiment, when using the reaction soln contain for example Tris damping fluid, EDTA etc., almost do not observe fluorescence.
Embodiment 3
In embodiment 3, confirmed after making nucleic acid and the copper that sputters at glass surface contacts, can detect fluorescence, and analyze the characteristic of fluorescence.
＜material and method 〉
As DNA, use the ssDNA that describes among the embodiment 1.As RNA, use the RNA that describes among the embodiment 2.
Employing is by ULVAC, Inc.(Kanagawn, Tokyo) SH-350 device sputter copper on glass surface that 99.99% Cu target (Kojundo Chemical Laboratory Co., Ltd., Saitama jade, Japan) is housed of making.In sputter, setting thickness is 40nm, based on the sedimentation velocity of measuring in advance, sets suitable sputtering time.The glass that is used for sputtering silver is the Kyodo International by Kanagawa, Japan, and Inc. produces.
In sputter on the glass slide or untreated glass slide of copper or silver, place sample solution, coverage gap cover glass (gap cover glass) (24 * 25No.4/#CG00024/Matsunami Glass Ind., Ltd., Osaka, Japan) thereon.After leaving standstill about 5 minutes, observe fluorescence.Inverted microscope Ti-U(Nikon Co., Tokyo, Japan) be used for observing.Bank of filters UV-1A(Ex:365/10, DM:400 BA:400/Nikon) is used for obtaining fluorescence.Numeral ccd video camera Retiga2000R(QImaging, BC, Canada) and 20 * object lens for catching and document image.
＜result 〉
Figure 22 is illustrated in the image that the sample of the NaCl of the DNA that contains 5mg/ml and 0.5M is caught after sputter has the glass surface of copper to leave standstill 5 minutes.Figure 23 is illustrated in the image that the sample of the NaCl of the RNA that contains 5mg/ml and 0.5M is caught after sputter has the glass surface of copper to leave standstill 5 minutes.
Shown in Figure 22 (A), when use contains the sample of DNA, observe the smoothly fluorescence of (smooth) at the whole image of catching.On the other hand, as Figure 23 (A) with (B), when use contains the sample of RNA, in the image of catching, observe the fluorescence with unique wavy pattern.The expectation reason of the peculiar pattern of RNA is that single stranded RNA hybridizes to generate higher structure each other.
Next, the fluorescence intensity in catching image is converted into numeral.Shown in Figure 22 (B), each is caught image and is divided into nine parts.One (C among the figure) in nine parts is set to useful range.Calculate the mean value of the fluorescence intensity in useful range.For each sample, catch five parts on the slide glass to calculate the mean value of each image.Standard deviation is calculated in the further mean deviation of five mean values of gained.
Figure 24 illustrate when the sample that contains DNA or RNA with sputter at copper on glass or silver when contacting, the fluorescence intensity of acquisition.In Figure 24, " DNA/Cu ", " RNA/Cu " and " (-)/Cu " expression contain the DNA of 5mg/ml sample, contain 5mg/ml RNA sample and do not contain the sample of nucleic acid; Fluorescence intensity is to have the glass surface of copper to measure in sputter.In addition, " DNA/Ag ", " RNA/Ag " and " (-)/Ag " expression contain the DNA of 5mg/ml sample, contain 5mg/ml RNA sample and do not contain the sample of nucleic acid; Fluorescence intensity is to measure at the glass surface of sputtering silver.Each sample contains the NaCl of 0.5M.Because the fluorescence intensity in " DNA/Cu " is apparently higher than other samples, its time shutter is 1 second.In all samples except " DNA/Cu ", the time shutter is 5 seconds.
As shown in the figure, in sputter the on glass of copper arranged, " DNA/Cu " and " RNA/Cu " has the fluorescence intensity that is higher than " (-)/Cu ".Especially in the DNA sample, detect strong fluorescence.On the other hand, on glass in sputtering silver compared with " (-)/Ag ", and " DNA/Ag " and " RNA/Ag " do not demonstrate the increase of fluorescence intensity.Compare with " (-)/Cu ", " (-)/Ag " shown higher observed value.This may be to be caused by the background from lip-deep reflected light, scattered light or the autofluorescence of sputtering silver.
Next, investigated the variation of fluorescence intensity with duration of contact of nucleic acid and copper.The sample that will contain the NaCl of the ssDNA of 5mg/ml and 0.5M is placed on sputter has the glass of copper and the time point between the gap cover slide to be designated as the starting point of measuring fluorescence intensity every the scheduled time.Caught image every 15 seconds, each acquisition phase opens and closes the exciting light shutter.Use * 10 object lens, the time shutter is 1 second.Each time, the piece image of catching is used to measure fluorescence intensity.The results are shown among Figure 25.
As shown in the figure, in the several minutes after introducing sample, fluorescence intensity increases gradually, and at about 3 minutes with the interior maximum value that reaches.
Contact with copper from nucleic acid, after one period scheduled time, investigate the variation of the fluorescence intensity that is caused by temperature variation.Catch after the image, immediately it is kept at room temperature.After 50 seconds, the heat block that is heated to 65 ℃ is placed on sputter lightly the on glass of copper.After 100 seconds, hot piece is removed.Caught image every 5 seconds.After 150 seconds, temporarily stop to measure and closing the exciting light shutter.After 900 seconds, begin again to measure.The results are shown among Figure 26.
As shown in the figure, in initial 50 seconds, fluorescence intensity reduces gradually.This may be caused by the fluorescence photofading.During ensuing 50 seconds, fluorescence disappears with the speed that obviously is different from the fluorescence photofading.After heat block was removed and gets back to room temperature, fluorescence recovered gradually.After 900 seconds, fluorescence intensity is got back to the level of the fluorescence intensity that deduction fades from initial fluorescent intensity.These results show from nucleic acid that copper contact the fluorescence launched be temperature-sensitive, and the reversible disappearance along with the temperature rising.
Embodiment 4
Embodiment 4 illustrates by celliferous sample is incorporated into sputter the on glass of copper, but the fluorescence observation nucleus.
＜material and method 〉
(Invitrogen Corporation, CA USA) are used as PBS to the Dulbecco phosphate buffered saline (PBS) of no Ca/Mg.
In the experiment of onion thin skin, with pair of forceps the thin skin of commercially available onion is carefully peeled off, be immersed in the distilled water and clean for use.The onion thin skin is positioned over sputter the on glass of copper, immerses among the PBS, covers with cover glass, and then observes.
In the experiment of human leucocyte sample, to IMMUNO-TROL Cells(Cat.No.6607077, Beckman Coulter, Inc., Fullerton, CA, USA) do following processing: at first, separate 500 milliliters of IMMUNO-TROL Cells, clean with PBS, and employing whizzer (1200rpm, 5 minutes) sedimentation.After this, discard supernatant liquid to peel off (flake) particle (pellet), repeat twice water-soluble blood and handle with sampling.Sample dilutes with PBS, prepares the white corpuscle sample thus.Water-soluble blood is handled and carried out as follows: after fully peeling off the precipitation that obtains as centrifugal product, add the 9ml deionized water, reversing mixed 30 seconds, 10 * PBS damping fluid (the Nippon Gene Co. that adds 1ml, Ltd., Tokyo, Japan) and fully stir.Cell is by centrifugation (1200rpm, 5 minutes), and sedimentation is to remove supernatant liquid.The white corpuscle sample is placed on sputter the on glass of copper, covers with cover glass, and then observes.
Sputter has identical among glass, cover glass, microscope of copper etc. and the embodiment 3.In sputter, thickness setting is 20nm, 40nm or 100nm.Except as otherwise noted, thickness setting is 40nm otherwise in following experiment.When a part of sputter copper on the glass slide surface only, paste the polyimide band on the glass slide surface, reserve the square of 5mm in the centre, thereby implement sputter.Remove the polyimide band then.Only like this, just be made into the sputter that has generated the Cu layer at the 5mm in centre square the glass of copper is arranged.
Adopt exciter filter (365/10nm), dichroscope (400nm) and fluorescence filters (590LP) that the onion thin skin is carried out Fluirescence observation.Adopt filter set UV-1A(Ex:365/10, DM:400, BA:400/Nikon) dialogue cell sample and Jurkat cell carry out Fluirescence observation.
＜result 〉
Figure 27 is illustrated in sputter the Fluirescence observation of onion thin skin on glass of copper and the image of catching.(a) and (b) be illustrated in the on glass observed image that sputter has copper.(c) and (d) not sputter is shown thereon and has observed image on the glass slide of copper.(a) and (c) be the observed image in bright field.(b) and (d) be fluoroscopic image.(a)～(d) be the image that adopts * 10 object lens to catch.(e) be the image that adopts * 40 object lens to catch.
As shown in the figure, there is the cell on glass of copper to observe the peculiar hyperfluorescence of nucleus in sputter.Although observe faint fluorescence at parts of fine cell wall etc., think the autofluorescence of cell walls etc., because it also is found on sputter not has cell on the glass slide of copper.
Next, observe zooblast.Figure 28 is illustrated in the Fluirescence observation and the image of catching that sputter has the human leucocyte sample on glass of copper.(a) be the observed image in bright field.(b) be fluoroscopic image.Used * 40 object lens.
In fluoroscopic image, obviously observe the peculiar paging nuclear of white corpuscle neutrophilic leukocyte (segmented neutrophils).
Figure 29 illustrates the observed image of copper sputtering glass that only uses at a part of sputter copper on glass slide surface.On the copper sputtering glass, scatter human leucocyte cell pearl, namely the Jurkat cell covers with cover glass, use then * 20 object lens observe.In the zone of deposited copper on the copper sputtering glass with there is not the border between the zone of deposited copper to catch image.(a) and (c) be the image that the bright field is observed; Occupy the black region that surpasses half and be can not printing opacity the zone because formed copper film.(b) and (d) be fluoroscopic image.
In the zone of deposited copper, only observe hyperfluorescence at the nucleus of cell.Figure 30 illustrates to use on it and generates thickness and be 20nm(a) or the copper sputtering glass of copper layer 100nm(b) observe the result of Jurkat cell.Under two kinds of thickness, all observe from nuclear fluorescence.
＜discuss
Result in the present embodiment shows by nucleus is contacted with copper, also can detect fluorescence.Obvious this phenomenon only occurs on it there to be on the glass substrate of copper sputter, and is the exercising result between nucleus and the copper.
Onion thin skin cell and leukocytic Fluirescence observation result obviously demonstrate the difference between the nuclear shape in each cell.Thus, according to the nucleic acid detection method of present technique, can identify the nuclear different shapes that depends on cell type.
Although do not illustrate in the present embodiment, but using on its part sputter to have in the experiment of glass slide of copper, after only observing the fluorescence from the cell on the zone of deposited copper, the slanted glass slide glass is so that cell moves to the not zone of deposited copper from the zone of deposited copper.After the movement, continue to observe fluorescence.Thus, even separate the position of the position of copper and cells contacting and Fluirescence observation cell, find to detect fluorescence by between two positions, placing the device that is used for migratory cell.
After the nuclear fluorescence that confirms from the cell between copper sputtering glass and cover glass, remove cover glass, celliferous solution is exposed in the air.Then, fluorescence rapidly disappears.Equally, in embodiment 1, use Cu(II) in the experiment of ion and S.A., to find to be exposed in the air after the long period at reaction soln, fluorescence disappears.The disappearance of fluorescence can be thought owing to contact the Cu(I that causes with air) oxidation of ion.Therefore, the generation (especially, being exposed to oxygen contained in the air) that may suppress fluorescence by sample solution is contacted with air.Think that be preferred by restriction and the contacting of air (for example in microchip) enforcement according to the nucleic acid detection method of present technique.
Embodiment 5
In embodiment 5, confirmed to embodiment 1 under the similar experiment condition, can be from having the oligomer DNA emitting fluorescence of two base length.
＜material and method 〉
Adopt with embodiment 1 in similar material and method, measured from the fluorescence of seven types oligomer DNA of Invitrogen company purchase.Base sequence in the oligomer DNA that uses is T(20) (sequence numbering: 1), T(10) (sequence numbering: 19), T(6) (sequence numbering: 10), T(5) (sequence numbering: 27), T(4) (sequence numbering: 28), T(3) (sequence numbering: 12), T(2) (sequence numbering: 29).Here, CuSO 4Concentration is set at 0.4mM, and S.A. concentration is set at 4mM, and NanoDrop3300 is used to measure.
＜result 〉
Figure 31 illustrates T(20) measuring result.The concentration separately of oligomer DNA is (a) 100 μ M, (b) 50 μ M, (c) 50 μ M, (d) 25 μ M, (e) 12.5 μ M and (f) 6.25 μ M.In each figure, transverse axis is represented wavelength (nm), and the longitudinal axis is represented fluorescence intensity (RFU value).Figure 32 to Figure 37 illustrates each oligomer DNA T(10), T(6), T(5), T(4), T(3) and measuring result T(2).The concentration of the numeric representation oligomer DNA among the figure shown in each curve.
Figure 38 is illustrated in the fluorescence spectrum that fluorescence intensity obtains for each oligomer DNA under the highest concentration conditions.Transverse axis is represented wavelength (nm), and the longitudinal axis is represented relative value (peak value RFU value is made as 1).(a) fluorescence spectrum expression T(20), (b) fluorescence spectrum expression T(10), (c) fluorescence spectrum expression T(6), (d) fluorescence spectrum expression T(5), (e) fluorescence spectrum expression T(4), (f) expression T(3) fluorescence spectrum, (g) expression T(2) fluorescence spectrum.
Figure 39 shows and has marked and drawed the figure of each oligomer DNA in the peak value RFU of each concentration value.Transverse axis is represented the concentration (μ M) of each oligomer DNA, and the longitudinal axis is represented peak value RFU value (logarithmic value).(a) expression T(20) the result, (b) expression T(10) the result, (c) expression T(6) the result, (d) expression T(5) the result, (e) expression T(4) the result, (f) expression T(3) the result, (g) expression T(2) the result.
＜discuss
The result of present embodiment has disclosed from the oligomer DNA with two base length of thymus pyrimidine and has observed fluorescence.Even when finding the concentration change when oligomer DNA, the shape of fluorescence spectrum is also almost constant, but fluorescence peak tends to shorten and move (with reference to Figure 38) to short wavelength side along with base length.As can be seen, the intensity of fluorescence spectrum tends to rely on the concentration of oligomer DNA, but when concentration surpasses a certain value, arrives maintenance level (plateau), and oppositely descend (with reference to Figure 39).And, in embodiment 2, use in the experiment of copper powder and observe, when the concentration of DNA was too high, fluorescence intensity was (with reference to the Figure 15) that reduces.
Embodiment 6
In embodiment 6, to embodiment 1 under the similar experiment condition, adopt the oligomer DNA with three base length that constituted by T and C or T and G to experimentize.
＜material and method 〉
Adopt with embodiment 1 in similar material and method, measure from the fluorescence of the oligomer DNA of Invitrogen Corporation purchase.12), TTC, TCT, CTT, TCC, CTC, CCT, CCC, TTG, TGT, GTT, TGG, GTG, GGT, GGG the sequence of used oligomer DNA is the TTT(sequence numbering:.CuSO 4Concentration is set at 0.4mM, and S.A. concentration is set at 4mM, and oligomer DNA concentration is set at 0.5mM, and NanoDrop3300 is used to measure.
＜result 〉
The results are shown in Figure 40 and 41.In Figure 40, (a) expression TTT(sequence numbering: result 12), (b) result of expression TTC, (c) result of expression TCT, (d) result of expression CTT, (e) result of expression TCC, (f) result of expression CTC, (g) represent the result of CCT, and (h) represent the result of CCC.In Figure 41, (a) expression TTT(sequence numbering: result 12), (b) result of expression TTG, (c) result of expression TGT, (d) result of expression GTT, (e) result of expression TGG, (f) result of expression GTG, (g) represent the result of GGT, and (h) represent the result of GGG.Transverse axis is represented wavelength (nm), and the longitudinal axis is represented the logarithmic value of fluorescence intensity (RFU value).
In the oligomer DNA of the mixed sequence with T and C, fluorescence intensity is the strongest in the TTT sequence, is CTT, CCT and TCT then.In TTC and CTC, define hypofluorescence (with reference to Figure 40).On the other hand, determine in TCC and CCC, not have at about 600nm place the fluorescence at peak.In the oligomer DNA of the mixed sequence with T and G, determined to have in the TTG sequence fluorescence of medium tenacity, at GTT hypofluorescence is arranged, but in other sequences, determine not have at about 600nm place the fluorescence (with reference to Figure 41) at peak.
＜discuss
In the oligomer DNA of the mixed sequence with T and C, show fluorescence intensity ratio TCT and TTC height second and the 3rd CTT sequence that comprises T.And, comprise that at the 3rd the TCT of T and CCT show that fluorescence intensity ratio is at second TTC and CTC height that comprises T.Can think that thus in the oligomer DNA of the mixed sequence with T and C, the fluorescence of the T in the 3rd base position is made very large contribution, and the fluorescence of the T in the second base position is made minor contributions.
In the oligomer DNA of the mixed sequence with T and G, except TTG and GTT, determine not have at about 600nm place the fluorescence at peak.Fluorescence intensity is lower than the oligomer DNA with mixed sequence that C and T are arranged usually.Thus, can think that G has the effect that absorbs fluorescent energy and quench fluorescence.
Embodiment 7
In embodiment 7, confirmed that fluorescence is by the cancellation of cancellation colorant.
＜material and method 〉
Adopt with embodiment 1 in similar material and method, the oligomer DNA T(10 that buys from Invitrogen Corporation) (sequence numbering: 19) and by Black Hole Quencher-2(BHQ2) modify oligomer DNA T(10) 3 ' end and oligomer DNA (T(10) BHQ2 that provides) (Sigma-Aldrich Corporation) be used to measure fluorescence.CuSO4 concentration is set at 0.4mM, and S.A. concentration is set at 4mM, and oligomer DNA concentration is set at 0.05mM, and NanoDrop3300 is used to measure.
＜result 〉
The results are shown among Figure 42.Transverse axis is represented wavelength (nm), and the longitudinal axis is represented fluorescence intensity (RFU value).At T(10) in observe tangible fluorescence, but at T(10 that quencher is modified) do not detect fluorescence among the BHQ2.
＜discuss
BHQ2 is the quencher of the light in the especially about 560nm of known effective absorption～650nm scope.Think to cause at T(10 owing to the effect of BHQ2) in the fluorescence observed at T(10) no longer can observe among the BHQ2.The result shows and the effect of copper can be combined with FRET.
Embodiment 8
In embodiment 8, fluorescence intensity and the spectral shape of thymus pyrimidine (T) and uridylic (U) are compared again, confirmed that intensity is different, but both spectral shapes are identical.In addition, the fluorescence of having investigated the cytosine(Cyt) that methylates (MeC) and inosine (I) produces, and has disclosed the two and all do not produce fluorescence.
＜material and method 〉
Adopt to embodiment 1 in similar material and method, measured the fluorescence of multiple oligomer DNA.The T(10 that buys from Invitrogen Corporation) (sequence numbering: 19), U(9) G(sequence numbering: 20), A(10) (sequence numbering: 30) and I(9) G(sequence numbering: 31) be used as oligomer DNA.And, the C(10 that buys from Sigma-Aldrich Corporation) (sequence numbering: 32), C(4) MeC(6) (sequence numbering: 33, MeC is 5-methyl 2-Deoxyribose cytidine) be used as oligomer DNA.CuSO 4Concentration is set at 0.4mM, and the concentration of S.A. is set at 4mM, and oligomer DNA concentration is set at 0.05mM, and NanoDrop3300 is used to measure.
＜result 〉
T(10) and U(9) each measuring result of three times is shown among Figure 43.(a) be the curve that has the X-coordinate of representative wavelength (nm) and represent the ordinate zou of fluorescence intensity (RFU value).(b) be to have representative as the curve of the ordinate zou of the mean value of the fluorescence intensity (RFU value) of relative value (the peak value RFU value of each oligomer DNA is set at 1).Confirmed U(9G) emission is than T(10) fluorescence that intensity is low, but have and T(10) similar spectral shape.
T(10), measuring result C(10) and C(4) MeC(6) is shown among Figure 44.Transverse axis represents wavelength (nm), and the longitudinal axis is represented fluorescence intensity (RFU).From T(10) observe tangible fluorescence, but from C(10) and C(4) MeC(6) do not observe fluorescence.
T(10), A(10) and I(9) measuring result of G is shown among Figure 45.Transverse axis represents wavelength (nm), and the longitudinal axis is represented fluorescence intensity (RFU).From T(10) observe tangible fluorescence, from A(10) observe hypofluorescence, but from I(9) do not observe fluorescence the G.
＜discuss
Compare with the fluorescence of launching from the nucleic acid with thymus pyrimidine sequence, the fluorescence intensity of launching from the nucleic acid with uridylic sequence is lower, but has similar spectral shape.Embodiment 1 supports this result.In addition, confirmed to have the sequence of cytosine(Cyt) or had cytosine(Cyt) and the nucleic acid emitting fluorescence not of the cytosine(Cyt) sequence that methylates.
These results show, by detecting fluorescence with copper, uridylic can be identified from cytosine(Cyt) and the cytosine(Cyt) that methylates.This shows according to nucleic acid detection method of the present invention can detect the uridylic (U) that undertaken by hydrosulfate reaction to the replacement of cytosine(Cyt) (C), and the methylating of analyzing DNA molecule.
Industrial applicibility
According to present technique, only by sample is contacted with copper, just can easy detection or measure whether nucleic acid exists and quantity, sample in base sequence; And shape, distribution, quantity and the size of cells in sample nuclear.
This technology is made contributions to improving nucleic acid in a plurality of fields that comprise medical field (pathology, tumor immunology, transplanting, genetics, regenerative medicine, chemotherapy etc.), drug discovery field, clinical examination field, field of food, agriculture field, engineering field, forensic science and criminal evaluation field or analysis and the analysis and research of cell.
Figure IDA00003140497900011
Figure IDA00003140497900021
Figure IDA00003140497900031
Figure IDA00003140497900041
Figure IDA00003140497900051
Figure IDA00003140497900061

Claims (10)

1. the detection method of a nucleic acid may further comprise the steps:
The sample that contains described nucleic acid is contacted with copper, and
Detection is from the fluorescence of described sample emission.
2. detection method according to claim 1, wherein,
Analyze the base sequence of described nucleic acid based on detected intensity of fluorescence and/or spectrum in the detection step.
3. detection method according to claim 1, wherein,
Analyze mispairing in the two strands that forms described nucleic acid based on detecting detected intensity of fluorescence in the step and/or spectrum.
4. detection method according to claim 1 may further comprise the steps:
Handle described sample with hydrosulfate, wherein,
The intensity of fluorescence that the intensity of fluorescence that goes out based on the described sample detection before hydrosulfate is handled in detecting step and/or spectrum and described sample detection after the hydrosulfate processing go out and/or the difference between the spectrum are analyzed methylating or demethylation of cytosine(Cyt) the described nucleic acid.
5. detection method according to claim 2, wherein,
Described copper is solid copper.
6. detection method according to claim 5, wherein,
Contact procedure is the step that under the coexistence of salt described sample is contacted with copper.
7. detection method according to claim 6, wherein,
Detecting step is to detect from the step of the fluorescence of described sample emission by the described sample of rayed with 300 μ m to 420 mum wavelengths.
8. optical observation method that contains the sample of nucleic acid may further comprise the steps:
Described sample is contacted with copper, and
Detection is from the fluorescence of described sample emission.
9. optical observation method according to claim 8, wherein,
Described sample is cell.
10. fluorescent substance, it comprises the mixture of copper and nucleic acid.
CN2011800532046A 2010-11-11 2011-11-04 Method for detecting nucleic acid, method for optically observing sample, and phosphor Pending CN103189509A (en)

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